Exosomes are small (30–100 nm) membrane vesicles secreted by a variety of cell types and only recently have emerged as a new avenue for cell-to-cell communication. They are natural shuttles of RNA and protein cargo, making them attractive as potential therapeutic delivery vehicles. MicroRNAs (miRNAs) are short non-coding RNAs which regulate biological processes and can be found in exosomes. Here we characterized the miRNA contents of exosomes derived from human neural stem cells (hNSCs). Our investigated hNSC line is a clonal, conditionally immortalized cell line, compliant with good manufacturing practice (GMP), and in clinical trials for stroke and critical limb ischemia in the UK (clinicaltrials.gov: NCT01151124, NCT02117635, and NCT01916369). By using next generation sequencing (NGS) technology we identified the presence of a variety of miRNAs in both exosomal and cellular preparations. Many of these miRNAs were enriched in exosomes indicating that cells specifically sort them for extracellular release. Although exosomes have been proven to contain miRNAs, the copy number quantification per exosome of a given miRNA remains unclear. Herein we quantified by real-time PCR a highly shuttled exosomal miRNA subtype (hsa-miR-1246) in order to assess its stoichiometry per exosome. Furthermore, we utilized an in vitro system to confirm its functional transfer by measuring the reduction in luciferase expression using a 3’ untranslated region dual luciferase reporter assay. In summary, NGS analysis allowed the identification of a unique set of hNSC derived exosomal miRNAs. Stoichiometry and functional transfer analysis of one of the most abundant identified miRNA, hsa-miR-1246, were measured to support biological relevance of exosomal miRNA delivery.

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Artificial tissues constructed from therapeutic cells offer a promising approach for improving the treatment of severe peripheral nerve injuries. In this study the effectiveness of using CTX0E03, a conditionally immortalised human neural stem cell line, as a source of allogeneic cells for constructing living artificial nerve repair tissue was tested. CTX0E03 cells were differentiated then combined with collagen to form engineered neural tissue (EngNT-CTX), stable aligned sheets of cellular hydrogel. EngNT-CTX sheets were delivered within collagen tubes to repair a 12 mm sciatic nerve injury model in athymic nude rats. Autologous nerve grafts (autografts) and empty tubes were used for comparison. After 8 weeks functional repair was assessed using electrophysiology. Further, detailed histological and electron microscopic analysis of the repaired nerves was performed. Results indicated that EngNT-CTX supported growth of neurites and vasculature through the injury site and facilitated reinnervation of the target muscle. These findings indicate for the first time that a clinically validated allogeneic neural stem cell line can be used to construct EngNT. This provides a potential ‘off the shelf’ tissue engineering solution for the treatment of nerve injury, overcoming the limitations associated with nerve autografts or the reliance on autologous cells for populating repair constructs.

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